Diagnosing Neurologic EHV-1: Back to Basics

Imagine the following scenario: Your horse has a fever. He's recently been exposed to a horse that tested positive for the neurologic strain of equine herpesvirus-1 (EHV-1). You call your vet, who comes out and swabs the horse's nasal passages and draws a tube or two of blood. A few days later you get a call confirming that your horse is positive for EHV-1. What exactly happened from the time your horse's samples left your barn to when you got that call?

Equine herpesvirus-1 is highly contagious and can cause a variety of ailments in horses, including rhinopneumonitis (a respiratory disease usually found in young horses), abortion in broodmares, and myeloencephalopathy (the neurologic form). Clinical signs of EHV-1 myeloencephalopathy include fever, ataxia (incoordination), weakness or paralysis of the hind limbs, and incontinence. The virus is generally passed from horse to horse via aerosol transmission (when affected animals sneeze/cough) and contact with nasal secretions, but it isn't transmissible to humans.

An ongoing outbreak of the virus affecting several Western states and Canadian provinces (believed to have started at the National Cutting Horse Association's [NCHA] Western Regional Championship competition that ended May 8) has many horse owners asking questions about all aspects of EHV-1. Udeni Balasuriya, BVSc, PhD, professor of virology at the University of Kentucky's Gluck Equine Research Center, and Nicola Pusterla, DVM, PhD, Dipl. ACVIM, an associate professor in the Department of Medicine and Epidemiology at the University of California, Davis, shared some insight on what happens when a horse's sample arrives at the laboratory, and what test results mean.

Infection or Exposure?

Researchers know that many horses are exposed to EHV-1 at a young age and latently carry (without showing illness) the virus with them throughout life. With this understood, are diagnostic tests able to differentiate between exposure and an active infection?

"The standard PCR assay detects viral nucleic acid in clinical specimens, but cannot differentiate active infection from latent EHV-1 infection in blood samples," Udeni Balasuriya, BVSc, PhD, professor of virology at the University of Kentucky's Gluck Equine Research Center, explained. "The problem is that while EHV-1 may be present in blood during clinical disease, it also becomes latent or dormant in some white blood cells." Thus, there is significantly more virus detected on PCR testing of a horse's blood during a febrile (fever) episode than when the virus is latent.

"Although real-time PCR cannot distinguish active infection from latent infection per se, it can calculate the viral copy number in clinical specimens," he added. "This type of real-time PCR assay--a quantitative real-time PCR (qPCR)--is used in Dr. Pusterla's (DVM, PhD, Dipl. ACVIM, an associate professor in the Department of Medicine and Epidemiology at the University of California, Davis) laboratory."

--Erica Larson

Sample Collection and Submission

"The collection and submission of appropriate clinical samples for laboratory diagnosis is very important," Balasuriya said, noting the importance of the veterinarian procuring both nasal swabs and blood during any suspected EHV-1 outbreak.

Nasal swabs and blood samples provide a chance at finding the virus itself or viral DNA (indicating whether the horse is shedding the virus), while serum samples afford an opportunity to detect the horse's antibodies to the virus.

Balasuriya explained that a veterinarian uses a cotton- or Rayon-tipped swab to collect a sample from the horse's nostril (The nose doesn't necessarily have to be runny--if the horse has been exposed to the virus, any secretion from the nasal passage will contain the virus). Afterward he or she places the swab immediately in viral transport media or sterile saline for its transport to the testing laboratory. Balasuriya noted that the swabs obtained from horses during early stages of EHV-1 respiratory disease--taken within the first five days of the onset of clinical signs--provide the best chance for detecting the live virus and/or viral DNA (yes, viruses have genes, too) using molecular biology techniques, such as polymerase chain reaction (PCR).

The veterinarian then collects blood samples in "vacutainer" tubes (the colored-top blood collection tubes that veterinarians often use), packs them on ice, and ships them overnight with the nasal swabs to the diagnostic lab.

The Ins and Outs of Diagnostic Testing

Once the samples arrive at the diagnostic lab, scientists conduct advanced diagnostic screenings to determine whether the horse is infected with EHV-1.

"The World Animal Health Organization (OIE) recommends standard PCR assay (test procedure) as one of the laboratory tests for the detection of EHV-1 in clinical specimens," Balasuriya said, adding that in most cases, a process called virus isolation should accompany PCR tests. Virus isolation shows whether the virus detected by PCR is alive or dead (which can indicate the horse's infection status; a dead virus indicates the horse has been exposed, but does not have an active infection), and it also allows the researchers to further characterize and study the virus. We'll break down each testing technique:

Polymerase chain reaction tests Both the nasal secretions collected on the swab and the blood samples can be tested using PCR, Balasuriya said. As mentioned before, PCR test identifies any viral DNA in the horse's nasal secretion or bloodstream. Balasuriya said, "The presence of virus (DNA) in nasal swabs shows the horse is actively shedding and is, therefore, a potential source of infection for all in-contact animals."

Also, "One of the reasons EHV-1, which is primarily a pathogen of the respiratory tract, can induce abortion or neurologic disease is that it has the ability to infect white blood cells and, thus, be transported throughout the body," Balysuriya explained. "High levels of EHV-1 in the blood are strongly correlated with clinical signs.

"Some laboratories use real-time PCR assays for the detection of EHV-1," he continued, explaining that this reliable diagnostic laboratory technique has a quick turnaround time, with results generally available within 24 hours. "This includes an allelic discrimination real-time PCR assay to distinguish between neuropathogenic (causing neurologic signs) and non-neuropathogenic strains of EHV-1," an assay that scientists recently developed in the Gluck laboratory where he works.

"This assay also has the capability to quantitate the virus load in clinical specimens (both nasal swabs and blood samples) if necessary," he added.

Blood Tests "Equine herpesvirus-1 can sometimes be detected indirectly by the presence of specific antibodies that are produced by the horse in response to infection (through blood tests)," Balasuriya said. "The current serological assays used in diagnostic laboratories detect antibodies (measured in titers) shared by both EHV-1 and EHV-4 and cannot distinguish between these two viruses."

Thus, Balasuriya suggested that two sequential blood samples are likely needed to provide a definitive diagnosis of EHV-1.

"Following vaccination or an infection, horses will usually produce antibodies to EHV-1, a process that takes a minimum of eight to 10 days," he said. "These antibodies will peak after 30 to 40 days and last for more than nine months. Consequently, serological testing of paired serum samples collected from a horse when clinical signs are first noticed, and again 21 to 28 days later can be useful as an additional procedure to assist in the diagnosis of EHV-1 infection during outbreaks." In other words, a veterinarian can compare the two antibody levels (at the onset of illness and during/after recovery) and make a diagnosis based on the quantitative difference between them.

Virus Isolation In some cases, after scientists have confirmed viral detection by PCR they examine the samples for the presence of a live virus by attempting to grow it in mammalian cell culture in the laboratory. As described briefly earlier, essentially virus isolation determines whether the DNA found during PCR is alive, indicating an active infection. If the scientist cannot isolate the virus, it indicates a latent infection or that the horse has simply been exposed and harbors the virus in an inactive form in the body. Additionally, virus isolation allows researchers the opportunity to learn more about the viruses affecting horses, offering perspective on what strains are appearing when and where.

"For research purposes, I think (virus isolation) is a must," Pusterla said. "Any organism linked to an infectious disease outbreak should at least be conserved and saved, otherwise we won't have the capability to update our vaccines (so they will be effective against particular strains). It's important for the veterinary community, and also for horse owners, to have these isolates ... to try to improve the health of the horses down the road if we consider updating vaccines. We gain information and understanding (about the virus).

Balasuriya concurred: "It is important to isolate EHV-1 strains from cases of equine myeloencephalopathy, abortion, and respiratory disease in cell culture for evaluation of their respective biological properties, such as antigenic variation (comparing that of the viral proteins among different strains) and virulence (capability of causing infection). In addition, molecular characterization of new isolates will allow us to better understand the emergence of new viral variants and evolution of EHV-1 in nature."

Identifying Mutations

"Previous research suggests a significant percentage of EHM outbreaks are caused by a ... variant strain," Balasuriya explained, noting that researchers previously discovered a single mutation in an EHV-1 gene that is associated with virus replication."However, there may be other mutations in the viral genome that may be associated with neurologic disease and this warrants further investigation."

"Recent studies clearly suggest that the development of (EHV-1) neurologic disease may be influenced by a variety of virus, host, and environmental factors," he continued. "Therefore, understanding the genetic basis of EHV-1 neurologic disease requires more basic research using molecular tools."

Pusterla explained that to identify these virus mutations, researchers will often grow an isolate of the virus in cell cultures. Then, when they have a large enough segment of viral DNA, they'll evaluate differences in their genome. He said, "These days, the entire genome of the virus can be determined. This can be compared to similar viruses from similar outbreaks. Everyone's talking about mutations and a super-virus now. We're dealing with a common virus that unfortunately is affecting a large population of horses. But this is a man-made situation (the transport and subsequent commingling of horses from different areas). We're not dealing with a new strain or anything like that. We're just dealing with the perfect storm (i.e., the situation at and after the NCHA show was ideal for the development of an outbreak)."

Finding the Source

With advanced diagnostic testing, will we ever be able to identify the lone horse responsible for spreading EHV-1 in the current outbreak in the western United States and Canada? In large-scale outbreaks--such as this one--it's not likely.

"By the time we realize we're dealing with an outbreak, we're at least seven or ten days away from when that horse--call it Typhoid Mary--spread the virus to other horses," Pusterla said. "You have, say, 800 new horses (all in one place). They're all healthy, otherwise they wouldn't be at that kind of performance level and competing. And that one horse doesn't need to display any clinical signs ... some people forget that. It's probable that the virus was brought in by a healthy-looking horse that ... exposed other horses to the virus."

Pusterla added that the veterinary community recognizes these so-called "subclinical shedders," or horses that appear healthy but are experiencing active infection, with the potential of shedding virus.

The origin of smaller-scale outbreaks are easier to pinpoint, he added.

"Let's say it happened on one small farm," he said. "There's a population of resident horses and a new horse comes in."

He explained that if the new horse isn't separated from the resident horses for an initial duration of about 21 days--and if the horse ends up being a subclinical shedder--new EHV-1 infections will likely be identified in the resident horses within seven to 10 days.

"In such a situation there is a good link between a possible subclinical shedder and resident horse exposure," he added.

Speaking of Links ...

Pusterla noted that EHV-1 outbreaks aren't necessarily uncommon in the United States: "Remember that EHV-1 outbreaks happen on a regular basis, but often on a smaller scale. The ongoing EHV-1 outbreak has involved many horses with several fatalities, which has gained more media attention. "

So could recent outbreaks reported in other U.S. regions be linked to the current one?

"Generally, they're probably not related," Pusterla said. "Right now, any horse developing fever or neurological signs that has a link to the Utah show is an EHV-1 suspect. Further, the outbreak is so far restricted to a specific breed, Quarter horses. Say I've got a Warmblood (testing positive) at the same time, 5,000 miles away. It's probably not linked because it's a different population of horses in a different geographic situation."

Take-Home Message

The process through which veterinarians diagnose EHV-1 is lengthy and labor-intensive. However, advances in scientific technology have allowed researchers to learn numerous things about a virus simply by testing a single nasal swab or blood sample. With each new wave of diagnostic assays, researchers are gaining a better understanding of neurologic EHV-1 and the genetics behind it. And, according to Balysuriya, ongoing research will help scientists develop effective vaccines against this important equine disease.

About the Author

Erica Larson, news editor, holds a degree in journalism with an external specialty in equine science from Michigan State University in East Lansing. A Massachusetts native, she grew up in the saddle and has dabbled in a variety of disciplines including foxhunting, saddle seat, and mounted games. Currently, Erica competes in eventing with her OTTB, Dorado.

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